Temed

Soft gel mixes contained: 550 µl of 7.6 mM hydrochloric acid (HCL), 330.5 µl of double-distilled water (ddH₂O), 0.5 µl N,N,N′,N′-tetramethylethylenediamine (TEMED) (Sigma), 20 µl 2% bis-acrylamide (BioRad), 70 µl of 40% acrylamide (BioRad), 20 µl 0.1 M NHS (N-hydroxysuccinimide, Sigma-Aldrich), 4 µl of 200 nm diameter beads resuspended at 0.2 µM (Invitrogen) and 5 µl of 10% ammonium persulfate (GE HealthCare) (prepared just before use). Stiff gels mixes contained: 550 µl of 7.6 mM HCL, 258.5 µl of ddH₂O, 0.5 µl of TEMED (Sigma), 25 µl 2% bis-acrylamide (BioRad), 137 µl of 40% acrylamide (BioRad), 20 µl of 0.1 M NHS (N-hydroxysuccinimide, Sigma-Aldrich), 4 µl of 200 nm diameter beads resuspended at 0.2 µM (Invitrogen) and 5 µl of 10% ammonium persulfate (GE HealthCare) (added just before use). A 12-μl drop of PAA mix was placed into the hydrophilic glass of a glass bottom dish (FD5040-100). The PAA mix was covered with a hydrophobic 13-mm diameter × 0.1 mm glass coverslips that were prepared fresh by coating them with PlusONE Repel-Silene ES (GE Healthcare) for 15 min at room temperature and dried with an air pistol. Polymerization proceeded for 45 min at room temperature in a humidifier chamber. The coverslip was carefully removed, and gels were washed three times for 2 min with 10 mM HEPES.

The protein samples from transfected cells and control were run on SDS-PAGE. The 15% acrylamide separating gel was prepared by adding 2.8 mL deionized water, 3 mL 40% acrylamide (Biorad, USA), 2 mL 1.5 M Tris pH 8.8 (Biorad, USA), 0.08 mL 10% SDS (Merck, Germany), 0.08 mL 10% APS (Sigma-Aldrich, Germany) and 0.008 mL TEMED (Merck, Germany). Then 4% stacking gel was prepared by adding 3.1 mL deionized water, 0.5 mL 40% acrylamide (Biorad, USA), 1.25 mL 0.5 M Tris pH 6.8 (Biorad, USA), 0.05 mL 10% SDS (Merck, Germany), 0.05 mL 10% APS (Sigma-Aldrich, Germany) and 0.005 mL TEMED (Merck, Germany). Finally, SDS-PAGE was started running at 100 V, 1.30 h.

To prepare PAH, acrylamide (with final concentrations of 3, 4, and 5%, Sigma), bis-acrylamide (0.1%, Severn Biotech), Ammonium persulfate (1%, Sigma), and TEMED (0.1%, Sigma) were dissolved in PBS. After mixing, around 40 μL of the solution was immediately directed into a tube (inner diameter 1 mm, Agilent Technologies) using a 1 mL syringe (BD plastic) connected to the other end of the tube and left there for 15 min at room temperature to cure. Then, the gel was expelled from the tube gently and at the same time was placed on the stretching device.
To prepare q-gel, parts A and B of q-Gel 920 (Quantum Silicones LLC), were mixed with ratios of 1:1.1, 1:2, and 1:3 and were transferred into a 30 × 3 × 3mm mould. After degassing the mixture, it was baked at 100 °C for 90min. Then the cured gel was removed from the mould carefully and mounted on the device. To prepare PDMS, the elastomer and curing agent (Ellsworth adhesives) were mixed with a ratio of 80:1, 10:1, and 5:1, and the mixture was moulded and degassed followed by baking at 90 °C for 90min. For the measurements, a very thin (~1mm) layer of the PDMS were peeled off from the bulk and placed on the stretching device.